The present invention relates generally to a novel receptor-type tyrosine kinase and to genetic sequences encoding same.
Tyrosine kinases form an important class of molecules involved in the regulation of growth and differentiation (1). One mode of proof for this role came from the identification of receptors which bind known soluble growth factors. The receptors for epidermal growth factor (EGF) (2), platelet derived growth factor (PDGF) (3) and colony stimulating factor-1 (CSF-1)(4) were all shown to be transmembrane molecules with the cytoplasmic regions encoding a tyrosine kinase catalytic domain. The CSF-1 receptor is homologous to the PDGF receptor in both the catalytic and extracellular domains (1,5). The extra cellular domain of these proteins is distinguished from other tyrosine kinases by the presence of immnunoglobulin-like repeats (1,6). Based on structural properties of the kinase domain, the c-kit protein was identified as another member of this family (7). The c-kit gene locus appears to underpin the defects in the congenitally anaemic W/W mouse (8-10). The ligand has now been identified (11-14) as shown to be encoded by the Sl locus. The locus is abnormal in the Steel mouse (15) which has identical defects to the W/W mouse but encodes a normal c-kit gene.
The other line of evidence for a critical role of tyrosine kinase proteins in growth control came from the study of viral oncogenes (16-17). These genes were shown to be directly involved in growth dysregulation by observations of a change in cell growth following introduction of DNA encoding these genes into fibroblasts. AU oncogenes have been shown to have close cellular homologues (proto-oncogenes). One of the first identified oncogenes was v-src, the cellular homologue (c-src) is the prototypical representative of the family of cytoplasmic tyrosine kinases which, following myristylation, become associated with the inner leaf of the cell membrane (18). Within the haemopoletic system a number of lineage-restricted src-like kinases have been defined (19).
The T cell-associated src-like kinase, lck, has been shown to associate independently with both the CD4 and CD8 transmembrane glycoproteins to form a signalling complex (20,21). By contrast, v-erb-B and v-fms, like their cellular homologues the EGF receptor and CSF 1 receptor, respectively, are transmembrane molecules encoding the entire signal transduction machinery in a single polypeptide (1,17).
Detailed analysis of the amino acid sequences of these proteins has revealed conserved structural motifs within the catalytic domains (5). Both tyrosine and serine-thereonine kinases have a consensus GXGXXG sequence (SEQ ID NO: 12) which is found in many nucleotide binding proteins (5). Other conserved sequence motifs are shared by both types of kinase while others are specific for the tyrosine or the threonine-serine kinase subgroups (5). The tyrosine kinases, while having regions of sequence conservation specific to this family, can be further subdivided according to the structural features of the regions 5xe2x80x2 to the catalytic domain (1,4-7). The novel tyrosine kinase of the present invention exhibits the same general characteristics as previously known tyrosine kinases.
In accordance with the present invention, a new receptor-type tyrosine kinase is provided and which is identified as a member of the eph/elk family of tyrosine kinases (22,23). The novel tyrosine kinase receptor is designated HEK (xe2x80x9chuman eph/elk-like kinasexe2x80x9d). As the present inventors have identified expression of HEK in both pre-B and T cell lines, the receptor molecule of the present invention and/or its ligand is contemplated herein to have particular applicability for use as agents in the in vivo modulation of the production and/or function of pre-B, B and T cells.
Accordingly, one aspect of the present invention provides an isolated receptor-type tyrosine kinase, said tyrosine kinase characterised by, in its naturally occurring form, being reactive to the monoclonal antibody III.A4, having an apparent molecular weight of approximately 120-150 kD in the glycosylated form and having an N-terminal amino acid sequence comprising:
E L I P Q P (SEQ ID NO: 1).
Preferably, the tyrosine kinase has an N-terminal amino acid sequence comprising:
(E L I P Q P S N E V N L X D (SEQ ID NO: 2),
wherein X is any amino acid and is preferably L.
More preferably, the tyrosine kinase has an N-terminal amino acid sequence comprising the amino acids:
E L I P Q P S N E V N L X D (S) K X1 I Q U (SEQ ID NO:3),
wherein X and X1 are any amino acid and preferably L and T, respectively.
Even more preferably, the tyrosine kinase comprises the amino acid sequence set forth in FIG. 1 or any parts or portions thereof, or having an amino acid sequence with at least 30% homology to the amino acid sequence set forth in FIG. 1 and having the identifying characteristics of HEK. More preferably, the degree of homology is at least 40%, still more preferably at least 55, even more preferably at least 70% and still even more preferably greater than 80%.
The hybridoma producing the monoclonal antibody III.A4 was deposited at Public Health Laboratory Service, European Collection of Animal Cell Cultures, Porton Down Salisbury, UK, on Jun. 20, 1991 under accession number 91061920.
The term xe2x80x9cisolatedxe2x80x9d as used in relation to the tyrosine kinase of the present invention includes a biologically pure preparation comprising at least 20%, preferably at least 40%, more preferably at least 60% and even more preferably at least 80% of the protein relative to other molecules as determined by weight, activity or other convenient means. The term also encompasses any form of the protein not in the naturally occurring state such as, but not limited to, a preparation of membranes containing the protein, a preparation of the protein separate from the membrane or a supernatant fluid comprising said protein. The preparation may be glycosylated, partially unglycosylated or complete unglycosylated or may have a glycosylation pattern altered from what is naturally occurring.
The tyrosine kinase of the present invention is expressed on a number of tumours of human origin. In particular, data are presented herein showing HEK expression in human lymphoid tumour cell lines LK63, Lila-1, JM, MOLT4 and HSB-2 and the human epithelial tumour HeLa. One skilled in the art, however, will immediately recognise that similar or homologous kinases may exist on non-tumour cells or on non-human tumours and which have similar properties to the tyrosine kinase of the present invention. For example, the results contained herein show some expression of HEK in heart muscle. Accordingly, the present invention extends to a tyrosine kinase functionally and structurally similar in any or all respects to the tyrosine kinase herein described including a kinase of non-tumour origin.
The present invention extends to preparations comprising the naturally occurring form of the tyrosine kinase protein, including any naturally occurring derivative forms thereof, as well as to synthetic and recombinant forms of the protein including any single or multiple amino acid substitutions, deletions and/or insertions to the polypeptide portion of the kinase and to analogues and homologues thereof. Such amino acid alterations to the molecule are examples of recombinant or synthetic mutants and derivatives of the kinase.
Insertions include amino acid and/or carboxyl terminal fusions as weU as intra-sequence insertions of single or multiple amino acids. Generally, insertions within the amino acid sequence will be smaller than amino or carboxyl terminal fusions, of the order of say 1 to 4 residues. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein. Deletional variants are characterised by the removal of one or more amino acids from the sequence. Substitutional variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place. Such subsitutions generally are made in accordance with the following Table 1.
Generally amino acids are replaced by other amino acids having like properties, such as hydrophobicity, hydrophilicity, electronegativity, bulky side chains, etc.
Amino acid substitutions are typically of single residues; insertions usually will be on the order of about 1-10 amino acid residues; and deletions will range from about 1-20 residues. Deletions or insertions preferably are made in adjacent pairs, i.e: a deletion of 2 residues or insertion of 2 residues.
The amino acid variants referred to above may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis (Merrifield; J. Am. Chem. Soc., 85: p2149, 1964) and the like, or by recombinant DNA manipulations. Techniques for making substitution mutations at predetermined sites in DNA having known sequence are weU known, for example M13 mutagenesis. The manipulation of DNA sequences to produce variant proteins which manifest as substitutional, insertional or deletional variants are well known in the art and are described for example in Maniatis et al (Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory, 1982).
Other examples of recombinant or synthetic mutants and derivatives of the tyrosine kinase protein of this invention include single or multiple substitutions, deletions and/or additions to any molecule associated with the kinase such as carbohydrates, lipids and/or proteins or polypeptides. Furthermore, it is possible that the tyrosine kinase protein of the present invention is a genetically altered version of a similar protein on normal cells. The present invention, therefore, extends to the ryrosine kinase protein from tumour or non-tumour origin and to all genetically altered forms thereof.
The terms xe2x80x9canaloguesxe2x80x9d and xe2x80x9cderivativesxe2x80x9d extend to any functional chemical equivalent of the tyrosine kinase protein characterised by its increased stability and/or efficacy in vivo or in vitro. The terms xe2x80x9canaloguexe2x80x9d and xe2x80x9cderivativesxe2x80x9d also extend to any amino acid derivative of the ryrosine kinase protein as described above.
Analogues of HEK contemplated herein include, but are not limited to, modifications to side chains, incorporation of unnatural amino acids and/or derivatising the molecule and the use of crosslinkers and other methods which impose conformational constraints on the peptides or their analogues. Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH4; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6 trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridbxylation of lysine with pyridoxal-5xe2x80x2-phosphate followed by reduction with NaBH4.
The guanidino group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitisation, for example, to a corresponding amide.
Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbomoylation with cyanate at alkaline pH.
Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hyroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
Modification of the imidaxole ring of a histidine residue may be accomplished by alkylation with jodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.
Examples of incorporating unnatural amino acids and derivatives during protein synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids.
Crosslinkers can be used, for example, to stabilise 3D conformations, using homo-bifunctional crosslinkers such as the bifunctional imido esters having (CH2)n spacer groups with n=1 to n=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bufinctional reagents which usually contain an amino-reactive moiety such as N-hydroxysuccinimide and another group specificreactive moiety such as maleimido or dithio moiety (SH) or carbodiimide (COOH). In addition, peptides could be conformationally constrained by, for example, incorporation of Cxcex1 and Nxcex1-methylamino acids, introduction of double bonds between Cxcex1 and Cxcex2 atoms of amino acids and the formation of cyclic peptides or analogues by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.
The present invention, therefore, extends to peptides or polypeptides and amino acid and/or chemical analogues thereof having the identifying characteristics of HEK as broadly described herein, and/or to regions thereof capable of, or responsible for, its action in transducing signals or in stimulating cellular responses such as growth and/or differentiation.
Accordingly, reference herein to the receptor-type tyrosine kinase of the present invention includes the naturally occurring molecule, recombinant, synthetic and analogue forms thereof and to any mutants, derivatives and human and non-human homologues thereof. All such kinases are encompassed by the term xe2x80x9cHEKxe2x80x9d.
The present invention further extends to the ligand for the novel receptor-type tyrosine kinase described herein and to any agonists and antagonists (e.g. soluble form of the receptor) of the enzyme. Since the tyrosine kinase is an oncogenic protein, antagonists to the receptor are of particular relevance and fall within the scope of the present invention. Such antagonists include antibodies (monoclonal and polyclonal), the enzyme itself in soluble form or otherwise, specific peptides, polypeptides or proteins and carbohydrates, amongst others. These types of antagonists are useful in developing anti-tumour agents where the growth or maintenance of the tumour itself is supported by the tyrosine kinase of the present invention. Accordingly, the addition of an effective amount of an antagonist to the tumour-associated receptor-type tyrosine kinase will inhibit, reduce or otherwise interfere with the receptor activity of the protein and thus prevent, reduce and/or inhibit tumour growth. The present invention, therefore, extends to pharmaceutical compositions comprising one or more antagonists to the tyrosine kinase herein described and one or more pharmaceutically acceptable carriers and/or diluents.
Ligand(s) for HEK are capable of being screened for in a number of ways. In one protocol, an expression vector (e.g. AP-TAG-HEK) is selected which encodes the entire extracellular region of HEK fused to an appropriate reporter molecule like alkaline phosphatase. The fusion protein expressed in cells is recovered from cell supernatants and used to stain (using the reporter molecule) tissue sections using the methods as described by Flanagan and Leder (39), the disclosure of which is incorporated herein by reference. Once cellular sources of ligand are identified these cells are then used to construct an expression library. If the ligand is cell bound (eg membrane bound), the expression vector (eg. AP-TAG-HEK) is used to stain pools to search for positive clones. If the HEK ligand is secreted, then another strategy will be required. In this case, supernatants of pools can be used to screen for induction of HEK phosphorylation in LK63 or HEK transfectants. Alternatively, supernatants from tissues producing HEK ligand can be used as a source in affinity purification on columns to which the product of, for example, pEE14-HEK is linked as a specific absorbent. The sequence of the purified ligand will be determined and this information used to clone the HEK ligand from cDNA libraries.
Another aspect of the present invention is directed to a nucleic acid isolate comprising a sequence of nucleotides encoding the novel receptor-type tyrosine kinase (including its recombinant, synthetic, mutant, derivative, analogue and homologue forms). The nucleic acid sequence may comprise deoxyribonucleotides or ribonucleotides and may exst in single or double stranded form, alone or in combination with a vector or expression vector molecule. The nucleic acid may be naturally occurring RNA or DNA or may be cDNA including complementary forms thereof. The nucleic acid molecule may also contain single or multiple nucleotide substitutions, deletions and/or additions relative to the nucleotide sequence encoding the naturally occurring or recombinant form of the protein. The vectors containing the nucleic acid sequences of the present invention may replicate in eukaryotes and/or prokaryotes and contain promoter sequences capable of expression in one or both of these types of cells. Suitable cells include mammalian, insect, yeast and/or bacterial cells. Particularly preferred cell types include CHO, baculovirus and E. coli cells. The preferred nucleotide sequence comprising HEK is set forth in FIG. 1. The general techniques of recombinant DNA technology, including isolation of recombinant proteins, are well known and are described for example in Maniatis et al (Supra).
This invention also provides a transgenic cell or cell culture carrying a nucleic acid isolate as described above.
In another aspect, this invention provides a pharmaceutical composition comprising a soluble form of the receptor-type tyrosine kinase as broadly described herein, said composition further comprising one or more pharmaceutically acceptable carriers and/or diluents.
This invention also extends to methods of use of the novel receptor-type tyrosine kinase of this invention and of antagonists to ligands binding to this tyrosine kinase.
In one aspect, this invention extends to a method of ameliorating the effects of interaction or binding between HEK and its ligand in a mammal comprising administering to said mammal an effective amount of the antagonist to a ligand binding to the tyrosine kinase of this invention.
The invention also extends to a method of phosphorylating a protein comprising contacting a preparation of said protein with an effective amount of the receptor-type tyrosine kinase of this invention for a time and under conditions sufficient to effect phosphorylation of the protein.
In yet another aspect, the invention provides a method of screening for a ligand bound to tissue or cells to the receptor-type tyrosine kinase of this invention comprising contacting the tyrosine kinase fused to a reporter molecule capable of producing a detectable signal to the tissue or cell sample to be tested for a time and under conditions sufficient for the fused tyrosine kinase to bind to a ligand on said tissue or cells and then detecting the reporter molecule.
The invention further provides a method of screening for a soluble ligand to the receptor-type tyrosine kinase of this invention comprising contacting a sample to be tested with a cell line capable of expressing the tyrosine kinase and screening for phosphorylation in said cell line.
One skilled in the art will, however, irtediately recognise that a variety of mutations, derivatives or chemical alternations can be made to the sequence to encode, for example, the analogues and derivatives disclosed above. The present invention also extends to short nucleic acid molecules which can act as nucleic acid probes to screen for the presence of the HEK gene or mutations therein.